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</br><A HREF="#scroll3">3. Cellular Uptaking</A>
</br><A HREF="#scroll3">3. Cellular Uptaking</A>
</br> <A HREF="#scroll4">4. PTEN Expression</A>
</br> <A HREF="#scroll4">4. PTEN Expression</A>
</br><ul id="list2"> <A HREF="#scroll4.1">4.1 Gene Expression</A></ul>
</br><ul id="list2"> <A HREF="#scroll4.1">4.1 Protein Expression</A></ul>
<ul id="list2"> <A HREF="#scroll4.2">4.2 Protein Expression</A></ul>
<ul id="list2"> <A HREF="#scroll4.2">4.2 Gene Expression</A></ul>
<A HREF="#scroll5">5. Cellular Viability</A>
<A HREF="#scroll5">5. Cellular Viability</A>
<A NAME="scroll1"></A></br> <A HREF="#scroll6">6. Conclusion</A>
<A NAME="scroll1"></A></br> <A HREF="#scroll6">6. Conclusion</A>
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<figure>
<figure>
</br><img src="http://openwetware.org/images/6/6b/1Fig5.png"height="246 " width="576"/>
</br><img src="http://openwetware.org/images/6/6b/1Fig5.png"height="246 " width="576"/>
<figcaption><font size="2">Figure 5: Fluorescence microscopy image of Block O structures located outside of cells.</font></figcaption>
<figcaption><font size="2">Figure 5: Block O and Branch Structures</font></figcaption>
</figure>
</figure>


Line 74: Line 74:
<figcaption><font size="2">Figure 6: TEM Images of Block O and Branch</font></figcaption>
<figcaption><font size="2">Figure 6: TEM Images of Block O and Branch</font></figcaption>
</figure>
</figure>
<h2>2. miR 21 Sequestering</h2>
<strong>Hypothesis:</strong> <i>Hypothesis: Structures are able to remove miR-21 from solution via complementary base pairing with miR-21 complementary overhangs.</i>
</br></br>In order to demonstrate the ability of the DNA nanostructures to sequester miR-21, structures functionalized with ssDNA overhangs complementary to miR-21 were incubated in solution at 37℃ with fluorescently-tagged miR-21 (Figure 7), purified via agarose gel electrophoresis, and subsequently analyzed by means of bulk fluorescence imaging. Sequestration of miR-21 is evident in sample bands when the fluorescence of the labeled miR-21 is present in the band containing our structures and fluorescence is diminished in the band containing free miR-21. A titration of miR-21 allowed us to determine the ability of the structures to sequester an increasing amount of free miR-21 before becoming saturated (Figure 8). As expected, when the control structure with scrambled overhangs was incubated with fluorescent miR there was no binding observed.


<figure>
<figure>
</br><img src="http://openwetware.org/images/d/dd/Fig7.png"height="246 " width="576"/>
</br><img src="http://openwetware.org/images/d/dd/Fig7.png"height="246 " width="576"/>
<figcaption><font size="2">Figure 7: TEM Images of Block O and Branch</font></figcaption>
<figcaption><font size="2">Figure 7: miR Sequestration Schematic</font></figcaption>
</figure>
</figure>
<figure>
<figure>
</br><img src="http://openwetware.org/images/d/d1/Fig8.png"height="246 " width="576"/>
</br><img src="http://openwetware.org/images/d/d1/Fig8.png"height="246 " width="576"/>
<figcaption><font size="2">Figure 8: TEM Images of Block O and Branch</font></figcaption>
<figcaption><font size="2">Figure 8: Bulk fluorescence image of a scrambled overhang control structure (c) incubated with a 3x excess of miR, functional structure incubated with a titration of miR</font></figcaption>
</figure>
</figure>
<div id="CellularUptaking">
<h2>3. Cellular Uptaking</h2>
</br><strong>Hypothesis:</strong><i>Our structures utilize the endolysosomal pathway to infiltrate cells.In addtion, the difference in size and aspect ratio of the structures will affect how they are uptaken</i>
</br></br>Once the structures were characterized and functionalized with miR-21 complementary overhangs, we sought to test whether they would be successfully uptaken into cells. One possible mode of uptake would be through endocytosis, where the structures would be enclosed in an endosome and allowed into the cell. To test this, structures labeled with a fluorescent intercalating dye (TOPRO3) were incubated with cells whose lysosomes were labeled with Lysotracker Green at a final concentration of 0.1 nM. After a 4 hour incubation period, the cells were imaged with a fluorescent microscope to observe colocalization between the signals originating from the structures and the lysosomes.


<figure>
<figure>
</br><img src="http://openwetware.org/images/a/a4/Fig9.png"height="246 " width="576"/>
</br><img src="http://openwetware.org/images/a/a4/Fig9.png"height="246 " width="576"/>
<figcaption><font size="2">Figure 9: TEM Images of Block O and Branch</font></figcaption>
<figcaption><font size="2">Figure 9: Fluorescent microscopy images exhibiting endosome (labeled in green) and DNA nanostructure (labeled in red) colocalization. Overlay of fluorescence signals with a DIC image is indicative of an endosomal cellular uptake mechanism.</font></figcaption>
</figure>
</figure>
</br>As shown in Figure 9, fluorescence from the Lysotracker (shown in green) and fluorescence from TOPRO3 (shown in red) occupy the same coordinates within a cell, suggesting that the structures and endosomes occupy the same space within the cell. In addition to suggesting a possible mechanism for uptake, the Lysotracker also aids in affirming the position of the structures within the cell. Since these images are two-dimensional, without the Lysotracker, there would be no way to determine whether the signal from the structures originated from within the cell, or from above or below the cell.
</br></br>One advantage of generating structures with very different aspect ratios was the ability to test which structures were more easily uptaken by cells. Although both structures fall within the ideal dimensional parameters for cell uptake[5], the different geometries of the elongated branch versus the compact Block O may have an impact on cell uptake efficiency[6]. While epifluorescent microscopy images do not necessarily provide quantitative data on the uptaking efficiency of structures, some preliminary assessments may be made nonetheless. Firstly, more instances of co-localization could be observed in the presence of branch structures relative to Block O structures. Perhaps more intriguingly, in the presence of branch, all instances of fluorescence in the 640 nm channel occurred either within viable cells or within dead cell debris following an incubation time of 4 hrs. However, a few instances of fluorescence were found outside of cells in the presence of Block O, as shown in Figure 10.


<figure>
<figure>
</br><img src="http://openwetware.org/images/e/e9/Fig10.png"height="246 " width="576"/>
</br><img src="http://openwetware.org/images/e/e9/Fig10.png"height="246 " width="576"/>
<figcaption><font size="2">Figure 10: TEM Images of Block O and Branch</font></figcaption>
<figcaption><font size="2">Figure 10: Fluorescent microscopy images exhibiting endosomes (green) and DNA nanostructure (red) colocalization. The fluorescence signal outside the cell in the Block O sample is indicative of slower uptake.</font></figcaption>
</figure>
</figure>


<figure>
</br>The fluorescence signal, with an intensity comparable to the intensity of the TOPRO3 dye, originated at the very edge of the cell, on the cell membrane. This may suggest that while branch structures are quickly uptaken once they come in contact with cells, the Block O structures experience a delay in uptake, and thus tend to congregate outside the cell. While this introduces an interesting possibility in terms of the mechanism of uptake, more experiments must be conducted both using qualitative methods such as fluorescent imaging, and quantitative methods such as qPCR to detect intracellular levels of DNA nanostructures to form a conclusion on this matter.
</br><img src="http://openwetware.org/images/0/07/Fig11.png"height="246 " width="576"/>
</div><!--end of cellular uptake-->
<figcaption><font size="2">Figure 11: TEM Images of Block O and Branch</font></figcaption>
</figure>
 
<h2>2. miR 21 Sequestering</h2>
<strong>Hypothesis:</strong> <i>Hypothesis: Structures are able to remove miR-21 from solution via complementary base pairing with miR-21 complementary overhangs.</i>


</br></br>In order to demonstrate the ability of the DNA nanostructures to sequester miR-21, structures functionalized with ssDNA overhangs complementary to miR-21 were incubated in solution at 37℃ with fluorescently-tagged miR-21 (Figure 7), purified via agarose gel electrophoresis, and subsequently analyzed by means of bulk fluorescence imaging. Sequestration of miR-21 is evident in sample bands when the fluorescence of the labeled miR-21 is present in the band containing our structures and fluorescence is diminished in the band containing free miR-21. A titration of miR-21 allowed us to determine the ability of the structures to sequester an increasing amount of free miR-21 before becoming saturated (Figure 8). As expected, when the control structure with scrambled overhangs was incubated with fluorescent miR there was no binding observed.




<h2>4. PTEN Expression</h2>
<h3>4.1 Protein Expression</h3>
</br><strong>Hypothesis:</strong><i>An increase in the relative expression of PTEN, a target protein for miR-21, will be observed after incubation of cells with our structures.</i>


<div id="CellularUptaking">
</br></br>The proof of successful structure uptake into cells allowed for the focus to shift to elucidating a possible mechanism through which the structures affect cell viability. As stated earlier, PTEN is a well established target of miR-21, and so it was hypothesized that any changes in miR-21 levels brought about by the structures will have an observable impact on relative PTEN protein levels within cells.
<h2>3. Cellular Uptaking</h2>
</br><strong>Hypothesis:</strong><i> Our structures utilize the endolysosomal pathway to infiltrate cells.</i>
</br></br>Once the structures were characterized and functionalized with miR-21 complementary overhangs, the next step was to test whether they would be successfully uptaken into cells. One possible mode of uptake would be through endocytosis, where the structures would be enclosed in an endosome and allowed into the cell. To test this theory, structures labeled with a fluorescent intercalating dye (TOPRO3) were incubated with cells whose lysosomes were labeled with Lysotracker Green. After a 4 hour incubation period, the cells were imaged with a fluorescent microscope to try and observe colocalization between the signals originating from the structures and the lysosomes.


</br></br> Cells were incubated with structures with complementary overhangs and scrambled overhangs at a final concentration of 0.1 nM for either 24 or 48 hrs. After the incubation time, total protein was extracted from the cells and a western blot was performed to compare the levels of PTEN between the treatment and the control. The levels of PTEN were normalized to GAPDH, a housekeeping protein, to control for sample and loading variability.
<figure>
<figure>
</br><img src="http://openwetware.org/images/2/25/CellularUptakingfig9.png"height="146 " width="576"/>
</br><img src="http://openwetware.org/images/0/07/Fig11.png"height="246 " width="576"/>
<figcaption><font size="2">Figure 3.1: Fluorescence microscopy image showing co-localization between structures and endosomes within cells. Top: Branch scrambled overhangs. Bottom: Block O scrambled overhangs.</font></figcaption>
<figcaption><font size="2">Figure 11: (a) Western blot image showing PTEN protein band (top) and GAPDH protein band (bottom) for different samples after a 24 hour (left) or 48 hour (right) incubation period. (b) Densitometric analysis showing normalized intensity relative to scrambled control for different samples at 24 hours (left) or 48 hours (right). The intensities of both PTEN and GAPDH bands were measured using ImageJ. The PTEN intensities were then divided by the corresponding GAPDH intensities to obtain a normalized value. The normalized intensities for each treatment (Branch miR, Block O miR) were normalized to its corresponding control (Branch scr, Block O scr) and plotted on a bar graph.</font></figcaption>
</figure>
</figure>
</br>As shown in Figure 9, fluorescence from the Lysotracker (shown in green) and fluorescence from TOPRO3 (shown in red) occupy the same coordinates within a cell, suggesting that the structures and endosomes occupy the same space within the cell. In addition to suggesting a possible mechanism for uptake, the Lysotracker also aids in affirming the position of the structures within the cell. Since these images are two-dimensional, without the Lysotracker, there would be no way to determine whether the signal from the structures originated from within the cell, or from above or below the cell. Co-localizing with Lysotracker affixes the signal’s position in the z-plane as well as the x and y planes.


</br></br>One advantage of generating structures with very different aspect ratios was the ability to test which structures were more easily uptaken by cells. Even though both structures meet the ideal dimensional parameters for cell uptake[22], the different geometries of the elongated branch versus the compact Block O may have an impact on cell uptake efficiency. While microscopy images do not necessarily provide quantitative data on the uptaking efficiency of structures, some preliminary assessments may be made nonetheless. Firstly, more instances of co-localization could be observed in the presence of branch structures relative to Block O structures. Perhaps more intriguingly, in the presence of branch, all instances of fluorescence in the 640 nm channel occurred either within viable cells or within dead cell debris. However, a few instances of fluorescence were found outside of cells in the presence of Block O, as shown in Figure 10.


</br></br>Figure 10
</br>The intensity of the bands on the blot, as shown in Figure 11, correspond to the level of protein expression in the sample. In samples incubated for 24 hours, densitometric analysis showed increased PTEN expression relative to the control for branch only. However, after 48 hours of incubation, an increase in PTEN expression was seen in both branch and Block O experimental samples.
</br>The fluorescence signal, with an intensity comparable to the intensity of the TOPRO3 dye, originated at the very edge of the cell, on the cell membrane. This may suggest that while branch structures are quickly uptaken once they come in contact with cells, the Block O structures experience a delay in uptake, and thus tend to congregate outside the cell. While this introduces an interesting possibility in terms of the mechanism of uptake, more experiments must be conducted both using qualitative methods such as fluorescent imaging, and quantitative methods such as qPCR to detect intracellular levels of DNA nanostructures to form a conclusion on this matter.
 
</div><!--end of cellular uptake-->
</br></br> This data reinforces our hypothesis by correlating the presence of miR-21 complementary structures to increased PTEN levels. It also reaffirms characteristics of the structures studies earlier, such as their ability to sequester miR-21, even within the cell environment. Of particular interest is the discrepancies between the behavior of the two structures. The samples incubated with Block O showed a delayed response to the treatment compared to the branch. One possible explanation for this observation is the lower efficiency of uptake for Block O seen in the cellular uptake experiment. If the Block O structures are indeed uptaken at a slower rate, it follows that they will reach the critical concentration necessary to facilitate change at a slower rate, resulting in the delayed response observed in this experiment. However, once uptaken, it is possible that the Block O structures are more effective at raising PTEN than branch is, since the Block O treatment resulted in a higher expression of PTEN compared to the branch treatment. This may be because the Block O is functionalized with more complementary overhangs than the branch, allowing it to induce a greater change per structure than the branch.
 
<h3>4.2 mRNA Expression</h3>
</br><strong>Hypothesis:</strong><i> Since miRNA functions by suppressing mRNA, a reduction in miRNA concentration by our structures should result in an increase in mRNA concentrations of PTEN, a miR-21 target gene</i>
 
</br></br>Another line of evidence for establishing a mechanism can be generated by studying changes in the relative level of the mRNA that codes for the target protein. mRNA quantification is usually carried out through a quantitative polymerase chain reaction (qPCR), which can detect the presence of minute quantities of target DNA with high specificity. The higher level of specificity allows for the detection of miniscule changes in mRNA levels that may not translate over to the protein level.
</br></br> Since mRNAs are the direct targets of miRNA, it was believed that changes in the mRNA level would be observed sooner than the resultant changes in the protein level. Therefore, incubation periods of 12 and 24 hours were chosen for analysis. As with protein expression experiments, the cells were incubated with structures at 0.1 nM final concentration for the selected time, and then lysed. However, instead of protein the total RNA from the cell was isolated, and subsequently converted to cDNA via reverse transcriptase PCR. qPCR was then performed on the samples with primers for PTEN and Actin, a housekeeping gene used to normalize the data.






<h2>4. PTEN Expression</h2>
<h3>4.1 Gene Expression</h3>
<h3>4.2 Protein Expression</h3>
<h2>5. Cellular Viability</h2>
<h2>5. Cellular Viability</h2>


Revision as of 19:50, 24 October 2014

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<h1>Project and Results</h1> <h2>Novel microRNA antisense therapeutic delivery using DNA origami nanostructures</h2>


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<TABLE BORDER="1" WIDTH="50%" CELLPADDING="4" CELLSPACING="3"> 

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<A HREF="#scroll1">1. Structures</A> </br> <A HREF="#scroll2">2. miR 21 Sequestering</A> </br><A HREF="#scroll3">3. Cellular Uptaking</A> </br> <A HREF="#scroll4">4. PTEN Expression</A> </br><ul id="list2"> <A HREF="#scroll4.1">4.1 Protein Expression</A></ul> <ul id="list2"> <A HREF="#scroll4.2">4.2 Gene Expression</A></ul> <A HREF="#scroll5">5. Cellular Viability</A> <A NAME="scroll1"></A></br> <A HREF="#scroll6">6. Conclusion</A> </br> <A HREF="#scroll7">7. Future Work</A>


</TD> 

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<h2>1. Structures</h2>

<strong>Hypothesis:</strong> <i>Combination of designed staples and scaffold DNA, in an optimized cationic solution will result in the synthesis of nanostructures with specific geometry.</i>

</br></br>Structures were designed to emulate and verify previous reports of ideal nanoparticle size for cellular uptake. It has been demonstrated that rod-shaped nanostructures, with sizes ranging from 20 to 120 nm, are uptaken more effectively than structures with a more cube-like shape[1]. Using this information, we designed two structures at opposite ends of the spectrum. The Block-O is essentially a torus, with outer dimensions of 30x30x24 nm, while the Buckeye Branch is a 90 nm long rod-like shape (Figure 5). Both structures were designed using caDNAno[2] and are able to incorporate ssDNA overhangs complementary to miR-21 (56 overhangs on The Block-O and 42 on the Branch). Three versions of each structure were designed; one with miR-21 complementary overhangs, a control with scrambled-sequence overhangs, and a structure without overhangs. Importantly, the scrambled overhangs were randomized so as to not create any epigenetic effects in the target.


<figure> </br><img src="http://openwetware.org/images/6/6b/1Fig5.png"height="246 " width="576"/> <figcaption><font size="2">Figure 5: Block O and Branch Structures</font></figcaption> </figure>


</br>To overcome the electrostatic repulsion of DNA oligomers, the addition of MgCl2 to the folding reaction (a solution containing ssDNA staple strands in a 10 fold molar excess relative to ssDNA scaffold strands, as well as a buffer containing EDTA and Tris) provides divalent cations which facilitating binding interactions. The concentration of these cations has been shown to have a pronounced effect on the yield and quality of folded structures[3]. To optimize this concentration, structures were folded with MgCl2 concentrations ranging from 12mM to 26mM. Subsequent analysis by means of both agarose gel electrophoresis and spectrophotometry showed that a concentration of 20mM MgCl2 provided the highest yields with the lowest concentration of misfolded structures. </br></br>The structures were further validated by direct imaging using Transmission Electron Microscopy (TEM), which showed branch structures with dimensions of 10 nm by 12 nm by 90 nm, and Block O structures with dimensions of 30 nm by 30 nm by 24 nm (Figure 6). This closely matches the designed dimensions. A small degree of global twist is evident in the branch, a result of inter-helical strain induced by the square helix pattern used in the design[4].

<figure> </br><img src="http://openwetware.org/images/9/99/Fig6.png"height="246 " width="576"/> <figcaption><font size="2">Figure 6: TEM Images of Block O and Branch</font></figcaption> </figure>

<h2>2. miR 21 Sequestering</h2> <strong>Hypothesis:</strong> <i>Hypothesis: Structures are able to remove miR-21 from solution via complementary base pairing with miR-21 complementary overhangs.</i>

</br></br>In order to demonstrate the ability of the DNA nanostructures to sequester miR-21, structures functionalized with ssDNA overhangs complementary to miR-21 were incubated in solution at 37℃ with fluorescently-tagged miR-21 (Figure 7), purified via agarose gel electrophoresis, and subsequently analyzed by means of bulk fluorescence imaging. Sequestration of miR-21 is evident in sample bands when the fluorescence of the labeled miR-21 is present in the band containing our structures and fluorescence is diminished in the band containing free miR-21. A titration of miR-21 allowed us to determine the ability of the structures to sequester an increasing amount of free miR-21 before becoming saturated (Figure 8). As expected, when the control structure with scrambled overhangs was incubated with fluorescent miR there was no binding observed.

<figure> </br><img src="http://openwetware.org/images/d/dd/Fig7.png"height="246 " width="576"/> <figcaption><font size="2">Figure 7: miR Sequestration Schematic</font></figcaption> </figure> <figure> </br><img src="http://openwetware.org/images/d/d1/Fig8.png"height="246 " width="576"/> <figcaption><font size="2">Figure 8: Bulk fluorescence image of a scrambled overhang control structure (c) incubated with a 3x excess of miR, functional structure incubated with a titration of miR</font></figcaption> </figure>


<div id="CellularUptaking"> <h2>3. Cellular Uptaking</h2> </br><strong>Hypothesis:</strong><i>Our structures utilize the endolysosomal pathway to infiltrate cells.In addtion, the difference in size and aspect ratio of the structures will affect how they are uptaken</i> </br></br>Once the structures were characterized and functionalized with miR-21 complementary overhangs, we sought to test whether they would be successfully uptaken into cells. One possible mode of uptake would be through endocytosis, where the structures would be enclosed in an endosome and allowed into the cell. To test this, structures labeled with a fluorescent intercalating dye (TOPRO3) were incubated with cells whose lysosomes were labeled with Lysotracker Green at a final concentration of 0.1 nM. After a 4 hour incubation period, the cells were imaged with a fluorescent microscope to observe colocalization between the signals originating from the structures and the lysosomes.

<figure> </br><img src="http://openwetware.org/images/a/a4/Fig9.png"height="246 " width="576"/> <figcaption><font size="2">Figure 9: Fluorescent microscopy images exhibiting endosome (labeled in green) and DNA nanostructure (labeled in red) colocalization. Overlay of fluorescence signals with a DIC image is indicative of an endosomal cellular uptake mechanism.</font></figcaption> </figure>

</br>As shown in Figure 9, fluorescence from the Lysotracker (shown in green) and fluorescence from TOPRO3 (shown in red) occupy the same coordinates within a cell, suggesting that the structures and endosomes occupy the same space within the cell. In addition to suggesting a possible mechanism for uptake, the Lysotracker also aids in affirming the position of the structures within the cell. Since these images are two-dimensional, without the Lysotracker, there would be no way to determine whether the signal from the structures originated from within the cell, or from above or below the cell.

</br></br>One advantage of generating structures with very different aspect ratios was the ability to test which structures were more easily uptaken by cells. Although both structures fall within the ideal dimensional parameters for cell uptake[5], the different geometries of the elongated branch versus the compact Block O may have an impact on cell uptake efficiency[6]. While epifluorescent microscopy images do not necessarily provide quantitative data on the uptaking efficiency of structures, some preliminary assessments may be made nonetheless. Firstly, more instances of co-localization could be observed in the presence of branch structures relative to Block O structures. Perhaps more intriguingly, in the presence of branch, all instances of fluorescence in the 640 nm channel occurred either within viable cells or within dead cell debris following an incubation time of 4 hrs. However, a few instances of fluorescence were found outside of cells in the presence of Block O, as shown in Figure 10.

<figure> </br><img src="http://openwetware.org/images/e/e9/Fig10.png"height="246 " width="576"/> <figcaption><font size="2">Figure 10: Fluorescent microscopy images exhibiting endosomes (green) and DNA nanostructure (red) colocalization. The fluorescence signal outside the cell in the Block O sample is indicative of slower uptake.</font></figcaption> </figure>

</br>The fluorescence signal, with an intensity comparable to the intensity of the TOPRO3 dye, originated at the very edge of the cell, on the cell membrane. This may suggest that while branch structures are quickly uptaken once they come in contact with cells, the Block O structures experience a delay in uptake, and thus tend to congregate outside the cell. While this introduces an interesting possibility in terms of the mechanism of uptake, more experiments must be conducted both using qualitative methods such as fluorescent imaging, and quantitative methods such as qPCR to detect intracellular levels of DNA nanostructures to form a conclusion on this matter. </div><!--end of cellular uptake-->


<h2>4. PTEN Expression</h2> <h3>4.1 Protein Expression</h3> </br><strong>Hypothesis:</strong><i>An increase in the relative expression of PTEN, a target protein for miR-21, will be observed after incubation of cells with our structures.</i>

</br></br>The proof of successful structure uptake into cells allowed for the focus to shift to elucidating a possible mechanism through which the structures affect cell viability. As stated earlier, PTEN is a well established target of miR-21, and so it was hypothesized that any changes in miR-21 levels brought about by the structures will have an observable impact on relative PTEN protein levels within cells.

</br></br> Cells were incubated with structures with complementary overhangs and scrambled overhangs at a final concentration of 0.1 nM for either 24 or 48 hrs. After the incubation time, total protein was extracted from the cells and a western blot was performed to compare the levels of PTEN between the treatment and the control. The levels of PTEN were normalized to GAPDH, a housekeeping protein, to control for sample and loading variability. <figure> </br><img src="http://openwetware.org/images/0/07/Fig11.png"height="246 " width="576"/> <figcaption><font size="2">Figure 11: (a) Western blot image showing PTEN protein band (top) and GAPDH protein band (bottom) for different samples after a 24 hour (left) or 48 hour (right) incubation period. (b) Densitometric analysis showing normalized intensity relative to scrambled control for different samples at 24 hours (left) or 48 hours (right). The intensities of both PTEN and GAPDH bands were measured using ImageJ. The PTEN intensities were then divided by the corresponding GAPDH intensities to obtain a normalized value. The normalized intensities for each treatment (Branch miR, Block O miR) were normalized to its corresponding control (Branch scr, Block O scr) and plotted on a bar graph.</font></figcaption> </figure>


</br>The intensity of the bands on the blot, as shown in Figure 11, correspond to the level of protein expression in the sample. In samples incubated for 24 hours, densitometric analysis showed increased PTEN expression relative to the control for branch only. However, after 48 hours of incubation, an increase in PTEN expression was seen in both branch and Block O experimental samples.

</br></br> This data reinforces our hypothesis by correlating the presence of miR-21 complementary structures to increased PTEN levels. It also reaffirms characteristics of the structures studies earlier, such as their ability to sequester miR-21, even within the cell environment. Of particular interest is the discrepancies between the behavior of the two structures. The samples incubated with Block O showed a delayed response to the treatment compared to the branch. One possible explanation for this observation is the lower efficiency of uptake for Block O seen in the cellular uptake experiment. If the Block O structures are indeed uptaken at a slower rate, it follows that they will reach the critical concentration necessary to facilitate change at a slower rate, resulting in the delayed response observed in this experiment. However, once uptaken, it is possible that the Block O structures are more effective at raising PTEN than branch is, since the Block O treatment resulted in a higher expression of PTEN compared to the branch treatment. This may be because the Block O is functionalized with more complementary overhangs than the branch, allowing it to induce a greater change per structure than the branch.

<h3>4.2 mRNA Expression</h3> </br><strong>Hypothesis:</strong><i> Since miRNA functions by suppressing mRNA, a reduction in miRNA concentration by our structures should result in an increase in mRNA concentrations of PTEN, a miR-21 target gene</i>

</br></br>Another line of evidence for establishing a mechanism can be generated by studying changes in the relative level of the mRNA that codes for the target protein. mRNA quantification is usually carried out through a quantitative polymerase chain reaction (qPCR), which can detect the presence of minute quantities of target DNA with high specificity. The higher level of specificity allows for the detection of miniscule changes in mRNA levels that may not translate over to the protein level. </br></br> Since mRNAs are the direct targets of miRNA, it was believed that changes in the mRNA level would be observed sooner than the resultant changes in the protein level. Therefore, incubation periods of 12 and 24 hours were chosen for analysis. As with protein expression experiments, the cells were incubated with structures at 0.1 nM final concentration for the selected time, and then lysed. However, instead of protein the total RNA from the cell was isolated, and subsequently converted to cDNA via reverse transcriptase PCR. qPCR was then performed on the samples with primers for PTEN and Actin, a housekeeping gene used to normalize the data.


<h2>5. Cellular Viability</h2>


<figure> </br><img src="http://openwetware.org/images/4/4b/5fig1.png"height="246 " width="476"/> <figcaption><font size="2"></font></figcaption> </figure> <figure> </br><img src="http://openwetware.org/images/4/4f/5fig2.png"height="246 " width="476"/> <figcaption><font size="2"></font></figcaption> </figure>

<figure> </br><img src="http://openwetware.org/images/8/8c/5table1.png"height="246 " width="476"/> <figcaption><font size="2"></font></figcaption> </figure>

<h2>6. Conclusion</h2> <h2>7. Future Work</h2> <br><a href="http://openwetware.org/index.php?title=Biomod/2014/OhioMOD/results&action=edit">Edit Project and Results</a><br><br> </div>


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